Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a wide variety of signal transduction processes in the cell. They have been shown to be key regulators in most cellular functions including proliferation, cell metabolism, cell survival, apoptosis, DNA damage repair, cell motility . . . . Uncontrolled signalling due to defective control of protein phosphorylation has been implicated in a number of diseases, including, for example, cancer, inflammation, allergies, immune diseases, CNS disorders, angiogenesis . . . .
Amongst the families of protein kinases, one particular example is the Adenosine Monophosphate-activated Protein Kinase (AMPK) family. Salt-Inducible Kinases (SIK) are part of the AMPKs, a family of serine/threonine protein kinases involved in highly conserved cascades that control these processes, and in particular play a role in cellular energy homeostasis. Three SIK isoforms have been identified, named SIK1 (also referred as SNF1-Like Kinase (SNF1LK) or Myocardial Snf1-like Kinase (MSK)), SIK2 (SNF1LK2 or KIAA0781) and SIK3 (KIAA0999) (Trends Endocrinol. Metab. (2004) 15: 21-26).
The cloning of SIK1 that was specifically expressed in the adrenal glands of high-salt diet-fed rats led to subsequent cloning of adipose-specific SIK2 and rather ubiquitous SIK3. SIK1 has a role in the fine-tuning of steroidogenic enzyme production during the initial phase of steroidogenesis (Mol. Endocrinol. (2001) 15: 1264-1276). SIK2 is induced by insulin, hormones, and differentiation factors in multiples cell types (adipocytes, neurons, macrophages, myocytes) and promotes cellular differentiation. This is related to the inhibition of CREB (cAMP Responsive Element Binding protein)-mediated gene expression, phosphorylation of Insulin Receptor Substrate-1 (IRS1), and activation of MEF2 (Myocyte Enhancer Factor 2)-mediated gene expression (J. Biol. Chem. (2003) 278: 18440-18447; Nature (2007) 449: 366-369; Proc Natl Acad Sci USA. (2012) 109: 16986-16991). SIK2 modulates the gene transcription through the phosphorylation of substrates leading to their nuclear export, such as the transcriptional activator complexes TORC (Transducer Of Regulated CREB activity) or the transcriptional inhibitor HDAC4 (Histone Deacetylase 4). Through a similar mechanism SIK3 induced chondrocyte differentiation (Development (2012) 139: 1153-1163). In drosophila, it was shown that SIK3 was upregulated in response to insulin and SIK3 mutant flies were sensitive to starvation, suggesting that SIK3 contributes to maintain energy balance being involved in the shift from glucose to fat burning under fasting conditions (Cell (2011) 145:596-606). SIK3 is also induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol (PLoS ONE (2012) 7: e37803). On the other hand, overexpression of SIK1 reduced hepatic triacylglycerol levels and lipogenic gene expression (The Journal of Biological Chemistry (2009) 284: 10446-10452). Thus, members of the SIK family are emerging as hormones and nutrients sensors, which modulate key transcriptional processes such as steroid hormone biosynthesis by the adrenal cortex, insulin signalling in adipocytes, or inflammatory cytokines in macrophages.
In context of cellular stresses associated with ATP-depletion, UV exposure, refeeding after starvation/ischemia, degradation of SIK kinases is rapidly induced after cAMP or calcium intracellular elevation. In respect to the cell types and SIK expressed proteins, these stresses activate the TORC-CREB-mediated gene expression and lead to a rapid stress cellular response. Hence, it was shown that inhibition of SIK2 after oxygen-glucose deprivation enhances neuron survival (Neuron (2011) 69:106-119) or promotes melanogenesis in melanoma cells (PLoS One (2011) 6:e26148).
Therapeutic strategies are needed to modulate the stress cellular response, such as during ischaemia and post reperfusion of tissue, in the chronic phase of cardiac remodelling, in diabetes and neurodegenerative conditions. The rapid activation or degradation of the SIK proteins, following multiple kinds of stresses, makes them interesting targets in inflammatory, cardiac or metabolic diseases and neurodegenerative disorders. SIK inhibition might also have application in cosmetology or pigmentation-related diseases to induce melanogenesis.
Besides the pivotal function in cellular energy homeostasis, the SIK proteins have also been involved in the regulation of the cell cycle. Inducible overexpression of SIK1 kinase domain in Chinese hamster ovary cells lead to cellular endoreplication (Genomics (2004) 83: 1105-1115). SIK2 plays a key role in the initiation of mitosis. It localizes at the centrosome where it phosphorylates the centrosome linker protein, C-Nap1, and its depletion blocked centrosome separation in mitosis (Cancer Cell (2010) 18: 109-121). Depletion of SIK2 also delayed G1/S transition and reduced the phosphorylation of AKT, a major protein associated with cell survival. This depletion blocked centrosome separation in mitosis, sensitizing ovarian cancers to paclitaxel in culture and in xenografts. Higher expression of SIK2 significantly correlated with poor survival in patients with high-grade serous ovarian cancers. We believe these data identify SIK2 as a plausible target for therapy in ovarian cancers. Moreover, expression of SIK3 was elevated in ovarian cancers, particularly in the serous subtype and at later stages. Overexpression of SIK3 in OVCAR3 cells promoted cell proliferation in culture and tumorigenicity following injection into nude mice (Oncogene (2011) 30: 3570-3584).
Taking into consideration the role of SIK proteins in the signalling pathways of nutrients and hormone sensors and of stress response, it was therefore an object of the present invention to provide a potent, selective, small molecule inhibitor of one or more of the SIK1, SIK2 or SIK3 which can block specifically SIK-dependent stress response or sensitize tumour cells to chemo or targeted therapies. By this means, it provides a therapeutic benefit in neurodegenerative disorders, pigmentation-related diseases and cancer as well as in cardiac, metabolic, autoimmune and inflammatory diseases characterized in increased stress responses and/or dysregulated SIK kinase activity; hereinafter referred to as ‘disorders associated with SIK kinase activity’.
We have now found that the macrocyclic pyrazolopyrimidines and imidazopyridazines and pharmaceutically acceptable compositions according to this invention are useful for the diagnosis, prevention and/or treatment of several disorders associated with SIK kinase activity (i.e. SIK-kinase associated diseases).